The present invention relates to a fluorescent substance using azamethine compounds which are useful as fluorescent labeling reagents and to its use. More particularly, the present invention relates to fluorescent nucleotides and fluorescent avidin using azamethine compounds, and uses thereof.
One of the most frequently used molecular biological techniques for detecting homologous nucleic acid sequences is DNA/DNA, RNA/RNA or RNA/DNA hybridization. In this technique, nucleic acid (DNA or RNA) used as a probe is labeled, and the labeled nucleic acid is hybridized to a nucleic acid (DNA or RNA) to be detected. When the nucleic acid used as a probe has a homology to the nucleic acid to be detected, each single-stranded nucleic acid hybridizes to its complementary sequence so as to form a double-stranded sequence, and then the double-stranded sequence is detected by a label of the probe.
Conventionally, when nucleic acid is used as a probe, a technique of labeling the probe with radioisotope has been employed and the presence of hybridization between the probe and a target nucleic acid has been detected using autoradiography.
Although the techniques using radioisotopes for labeling a gene probe is especially superior in its high sensitivity, there exist such problems that the handling of radioisotopes is complicated because safety of the laboratory must be ensured and special care must be taken in the disposal of radioactive wastes. Moreover, radioisotopes can be used only for a limited time because they have a half-life period.
Therefore, non-radioactive labeling techniques have been developed as more simple techniques. For example, techniques of labeling a gene probe with biotin molecules (European Patent No. 0 063 879) or with digoxigenin molecules (European Patent Application No. 0 324 474 A1) are known. After hybridization of a labeled nucleic acid probe to the nucleic acid sequence to be detected, biotin molecules or digoxigenin molecules are present in the resulting double-stranded nucleic acid. After hybridization, binding of (strept)avidin-marker enzyme complex or anti-digoxigenin antibody-marker enzyme complex to the resultant double-stranded nucleic acid sequence allows detection of nucleic acids to which the probes was hybridized. However, such detection methods using enzymes are disadvantageous in terms of sensitivity and specificity.
Other than the above techniques, various techniques of labeling a target substance with fluorescent dye have been studied. For example, a desired fluorescent labeling reagent (1) possesses a high fluorescent quantum yield, (2) possesses a high molecular extinction coefficient, (3) is water-soluble and does not self-quench by agglutinating in an aqueous solvent, (4) is not susceptible to hydrolysis, (5) does not photo-dissociate easily, (6) is not susceptible to background fluorescence, and (7) has a previously introduced reactive substituent which forms covalent bonding with a target substance.
Fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate, which are well-known as fluorescent labeling reagents, possess high fluorescent quantum yields, but have drawbacks such that the molecular extinction coefficients is low and the excitation and luminous wavelength is 500 nm to 600 nm and therefore these reagents are susceptible to the influence of background fluorescence of a membrane used for blotting.
As dyes having a high molecular extinction coefficient, polymethine dyes are known such as cyanine dye described in U.S. Pat. No. 5,486,616, Japanese Patent Application Laid-Open Nos. 2-191674, 5-287209, 5-287266, 8-47400, 9-127115, 7-145148 and 6-222059, and barbiturate oxonol described in Journal of Fluorescence, 5, 231, 1995. However, there exist some problems such that they are almost insoluble in water and if they are dissolved, hydrolysis occurs. Also, strong intermolecular interactions between dyes can cause formation of aggregates in an aqueous medium so that self-quenching of fluorescence is often observed.
Moreover, cyanine dyes described in Japanese Patent Application Laid Open No. 2-191674 and the like are superior dyes because they have water-solubility due to introduction of a sulfonic acid group into a relatively stable chromophore and the formation of aggregates is prevented. However, its fluorescent quantum yield is not sufficiently high and synthesis of the dye is difficult due to the introduction of a sulfonic acid group. Under such circumstances, it has been required to develop a fluorescent dye which has a strong fluorescence as well as a high water-solubility and stability enough to cause no quenching of fluorescence due to aggregation.
Another example of a frame dye with high fluorescence intensity is azaindoleninecyanin dye described in GB Patent No. 870,753.However, applicability of this azaindoleninecyanin dye as a fluorescent labeling reagent remains unknown because in this patent application, there is no description as to essential features of a fluorescent labeling reagent, such as water-solubility, cohesiveness and solution stability, and there is no examples of introduction of a reactive substituent which causes covalent bonding with a target substance. Moreover, examples of application of azaindoleninecyanin to photographic use are shown in Japanese Patent Application Laid Open Nos. 4-358143, 3-135553, 1-280750 and European Patent No. 341958. These examples utilize absorbance characteristics of azaindoleninecyanin, but do not focused its luminescent characteristics and do not positively utilize it.
The object to be solved by the invention is to overcome the abovementioned problems of the conventional techniques. The object to be solved by the present invention is to provide a novel fluorescent nucleotide which is useful in labeling nucleic acid, and a novel fluorescent avidin which is useful in analyzing biological components such as nucleic acids, proteins or sugars.
Having conducted intensive study to solve the above described problems, the present inventors formed a complex of a nucleotide and a recently developed fluorescent labeling reagent, i.e. an azamethine compound, and labeled and detected nucleic acids using this complex. As a result, the inventors have found that the complex is superior in the ratio of uptake into nucleic acids and the fluorescent intensity in the detection. Furthermore, the present inventors formed a conjugate of a streptavidin and an azamethine compound, and detected biotin-labeled nucleic acids using the conjugate. As a result, the inventors found that the fluorescence is detected in an intensity which depends on the level of nucleic acid. The present invention has been completed on the basis of these findings.
According to the present invention, there is provided a fluorescent substance which is represented by a formula: A-B-C
wherein
A is a residue of natural or synthetic nucleotide, oligonucleotide, polynucleotide, or derivative thereof, and binds to B at a base moiety in said residue, or A is a residue of avidin or streptavidin;
B is a divalent linking group or a single bond; and
C is a monovalent group derived from a general formula (I) and binds to B at a reactive group present in R1 or R2: 
wherein R1 and R2 each independently represent an alkyl group that may be substituted with a reactive group capable of covalently bonding to A-B-; R3, R4, R5, and R6 each independently represent an alkyl group, and R3 and R4, and/or R5 and R6 may bind to each other to form a saturated carbon-ring together with a carbon atom(s) to which they bind; V1, V2, V3, V4, V5, V6, V7, V8, V9 and V10 each independently represent a hydrogen atom or a monovalent substituent, and two adjacent groups thereof may bind to form a ring; L1, L2, and L3 represent a substituted or unsubstituted methine group; each of m, n, s, and t represents 0 or 1, provided that m+n=1 and s+t=1; p represents 1, 2, or 3; M represents a counter ion; and q represents a number required to neutralize the charge of a molecule.
Preferably, at least one of R1 and R2 is an alkyl group substituted with an active ester group capable of covalently bonding to an amino group, a hydroxyl group or a thiol group which is present in the group represented by A-B-.
Preferably, at least one of R1 and R2 is an alkyl group substituted with a carboxyl group.
Preferably, A is a residue of natural or synthetic nucleotide, oligonucleotide, or polynucleotide or derivative thereof.
Preferably, A is a residue of nucleotide or derivative thereof.
Preferably, A is a residue of natural or synthetic nucleotide or derivative thereof, which are selected from the group consisting of: (1) nucleotides consisting of AMP, ADP, ATP, GMP, GDP, GTP, CMP, CDP, CTP, UMP, UDP UTP, TMP, TDP, TTP, 2-Me-AMP, 2-Me-ADP, 2-Me-ATP, 1-Me-GMP, 1-Me-GDP, 1-Me-GTP, 5-Me-CMP, 5-Me-CDP, 5-Me-CTP, 5-MeO-CMP, 5-MeO-CDP, and 5-MeO-CTP; (2) deoxynucleotides and dideoxynucleotides corresponding to said nucleotides; and (3) derivatives derived from said nucleotides as described in (1) and (2).
Preferably, A is a residue of avidin or streptavidin.
Preferably, B is a linking group selected from xe2x80x94CH2xe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94C. Cxe2x80x94, xe2x80x94COxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94 or combinations thereof, wherein a hydrogen atom on the linking group may be further substituted with a substituent.
Preferably, B is an aminoallyl group.
According to another aspect of the present invention, there is provided a process of preparing fluorescence-labeled nucleic acids which comprises the step of conducting a reaction of the synthesis of nucleic acid by using nucleic acid synthetase, a nucleic acid as a template, and the fluorescent substance of the invention.
Preferably, the reaction of the synthesis of nucleic acid is a reaction selected from the group consisting of a reverse transcription reaction, a terminal transferase reaction, a random prime method, a PCR method, and a nick-translation method.
According to further another aspect of the present invention, there is provided a nucleic acid probe or primer which is labeled with the fluorescent substance according to the invention.
According to still further another aspect of the present invention, there is provided a diagnostic agent or a reagent for detecting nucleic acids, which comprises the fluorescent substance according to the invention.
According to still further another aspect of the present invention, there is provided a kit for detecting nucleic acids, which comprises (1) the fluorescent substance according to the invention, (2) a nucleic acid synthetase, and (3) a buffer.
According to still further another aspect of the present invention, there is provided an analytical reagent or a diagnostic agent consisting of the fluorescent substance according to the invention.
According to still further another aspect of the present invention, there is provided an analytical kit, which comprises (1) the fluorescent substance according to the invention, and (2) biotin or a biotin-labeled substance.